Complexes of signaling proteins that are nucleated upon activation of receptor tyrosine kinases are dynamic macromolecular assemblies held together by interactions, such as the recognition of phosphotyrosines by Src homology 2 (SH2) domains. We predicted that reversible binding and phosphatase activity enable dynamic regulation of these protein complexes, which could affect signal transduction. We explored how dynamics in the interactions among the epidermal growth factor (EGF) receptor (EGFR), GRB2-associated binder protein 1 (GAB1), and SH2 domain-containing phosphatase 2 (SHP2) affected EGFR signaling output, specifically SHP2 binding to tyrosine-phosphorylated GAB1, which relieves the autoinhibition of SHP2. Among the effects of activated SHP2 is increased extracellular signal-regulated kinase (ERK) activity. We found that in H1666 lung adenocarcinoma cells, EGFR-activated Src family kinases (SFKs) counteracted repeated GAB1 dephosphorylation events and maintained the association of SHP2 with phosphorylated GAB1 at a cytosolic site distal from EGFR. A computational model predicted that an experimentally verified delay in SFK inactivation after EGFR inactivation, combined with an amplification of GAB1 phosphorylation in cells with proteins in a specific range of concentrations, enabled GAB1 phosphorylation and GAB1-SHP2 complexes to persist longer than EGFR phosphorylation persisted in response to EGF. This SFK-dependent mechanism was specific to EGFR and did not occur in response to activation of the receptor tyrosine kinase c-MET. Thus, our results quantitatively describe a regulatory mechanism used by some receptor tyrosine kinases to remotely control the duration of a signal by regulating the persistence of a signaling protein complex.

Non-small cell lung cancer (NSCLC) cells harboring activating mutations of the epidermal growth factor receptor (EGFR) tend to display elevated activity of several survival signaling pathways. Surprisingly, these mutations also correlate with reduced phosphorylation of ERK and SHP2, a protein tyrosine phosphatase required for complete ERK activation downstream of most receptor tyrosine kinases. Since ERK activity influences cellular response to EGFR inhibition, altered SHP2 function could play a role in the striking response to gefitinib witnessed with EGFR mutation. Here, we demonstrate that impaired SHP2 phosphorylation correlates with diminished SHP2 function in NSCLC cells expressing mutant, versus wild-type, EGFR. In NSCLC cells expressing wild-type EGFR, SHP2 knockdown decreased ERK phosphorylation, basally and in response to gefitinib, and increased cellular sensitivity to gefitinib. In cells expressing EGFR mutants, these effects of SHP2 knockdown were less substantial, but expression of constitutively active SHP2 reduced cellular sensitivity to gefitinib. In cells expressing EGFR mutants, which do not undergo efficient ligand-mediated endocytosis, SHP2 was basally associated with GAB1 and EGFR, and SHP2′s presence in membrane fractions was dependent on EGFR activity. Whereas EGF promoted a more uniform intracellular distribution of initially centrally localized SHP2 in cells expressing wild-type EGFR, SHP2 was basally evenly distributed and did not redistribute in response to EGF in cells with EGFR mutation. Thus, EGFR mutation may promote association of a fraction of SHP2 at the plasma membrane with adapters which promote SHP2 activity. Consistent with this, SHP2 immunoprecipitated from cells with EGFR mutation was active, and EGF treatment did not change this activity. Overall, our data suggest that a fraction of SHP2 is sequestered at the plasma membrane in cells with EGFR mutation in a way that impedes SHP2′s ability to promote ERK activity and identify SHP2 as a potential target for co-inhibition with EGFR in NSCLC.

The tyrosine phosphorylated epidermal growth factor receptor (EGFR) initiates numerous cell signaling pathways. Although EGFR phosphorylation levels are ultimately determined by the balance of receptor kinase and protein tyrosine phosphatase (PTP) activities, the kinetics of EGFR dephosphorylation are not well understood. Previous models of EGFR signaling have generally neglected PTP activity or computed PTP activity by considering data that do not fully reveal the kinetics and compartmentalization of EGFR dephosphorylation. We developed a compartmentalized, mechanistic model to elucidate the kinetics of EGFR dephosphorylation and the coupling of this process to phosphorylation-dependent EGFR endocytosis. Model regression against data from HeLa cells for EGFR phosphorylation response to EGFR activation, PTP inhibition, and EGFR kinase inhibition led to the conclusion that EGFR dephosphorylation occurs at the plasma membrane and in the cell interior with a timescale that is smaller than that for ligand-mediated EGFR endocytosis. The model further predicted that sufficiently rapid dephosphorylation of EGFR at the plasma membrane could potentially impede EGFR endocytosis, consistent with recent experimental findings. Overall, our results suggest that PTPs regulate multiple receptor-level phenomena via their action at the plasma membrane and cell interior and point to new possibilities for targeting PTPs for modulation of EGFR dynamics.

Information from multiple signaling axes is integrated in the determination of cellular phenotypes. Here, we demonstrate this aspect of cellular decision making in glioblastoma multiforme (GBM) cells by investigating the multivariate signaling regulatory functions of the protein tyrosine phosphatase SHP2. Specifically, we demonstrate that SHP2's ability to simultaneously drive ERK and antagonize STAT3 pathway activities produces qualitatively different effects on the phenotypes of proliferation and resistance to EGFR and c-MET co-inhibition. While the ERK and STAT3 pathways independently promote proliferation and resistance to EGFR and c-MET co-inhibition, SHP2-driven ERK activity is dominant in driving cellular proliferation, and SHP2's antagonism of STAT3 phosphorylation prevails in promoting GBM cell death in response to EGFR and c-MET co-inhibition. Interestingly, the extent of these SHP2 signaling regulatory functions is diminished in glioblastoma cells expressing sufficiently high levels of the EGFR variant III (EGFRvIII) mutant, which is commonly expressed in GBM. In cells and tumors expressing EGFRvIII, SHP2 also antagonizes EGFRvIII and c-MET phosphorylation and drives expression of HIF-1/2α, adding complexity to the evolving understanding of SHP2's regulatory functions in GBM.

Phosphorylation (activation) and dephosphorylation (deactivation) of the slit diaphragm proteins NEPHRIN and NEPH1 are critical for maintaining the kidney epithelial podocyte actin cytoskeleton and, therefore, proper glomerular filtration. However, the mechanisms underlying these events remain largely unknown. Here we show that NEPHRIN and NEPH1 are novel receptor proteins for hepatocyte growth factor (HGF) and can be phosphorylated independently of the mesenchymal epithelial transition receptor in a ligand-dependent fashion through engagement of their extracellular domains by HGF. Furthermore, we demonstrate SH2 domain–containing protein tyrosine phosphatase-2–dependent dephosphorylation of these proteins. To establish HGF as a ligand, purified baculovirus-expressed NEPHRIN and NEPH1 recombinant proteins were used in surface plasma resonance binding experiments. We report high-affinity interactions of NEPHRIN and NEPH1 with HGF, although NEPHRIN binding was 20-fold higher than that of NEPH1. In addition, using molecular modeling we constructed peptides that were used to map specific HGF-binding regions in the extracellular domains of NEPHRIN and NEPH1. Finally, using an in vitro model of cultured podocytes and an ex vivo model of Drosophila nephrocytes, as well as chemically induced injury models, we demonstrated that HGF-induced phosphorylation of NEPHRIN and NEPH1 is centrally involved in podocyte repair. Taken together, this is the first study demonstrating a receptor-based function for NEPHRIN and NEPH1. This has important biological and clinical implications for the repair of injured podocytes and the maintenance of podocyte integrity.